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Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group (1-13) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3, 5, 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC50 values were in the range of 22.99-51.66 µM (HCT 116 cell line), 8.63-41.20 µM (BxPC-3 cell line) and 24.78-81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line (p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1, 2, 3 and 5) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.

This study aimed to determine the phenolic compound content, in vitro antioxidative potential, and cytotoxic effects of four red wine samples: a commercial (V) and three clonal wines (V1, V2, and V3). LC/MS-MS, cyclic voltammetry, and MTT assay techniques were employed for this purpose. Results revealed that all wines were rich in phenolic compounds. Clonal wines outperformed the commercial ones in most phenolic compounds (except myricetin). Notably, V2 and V3 showed the highest levels of gallic acid, catechin, and epicatechin. Among them, V3 exhibited superior antioxidative activity. The MTT assay demonstrated stronger cytotoxic effects of the wine samples on pancreas (Bx-PC3) and colon (HT29) carcinoma cells (47% to 16% and 27% to 7% compared to control, respectively) than on the normal lung fibroblasts (MRC-5) cell line (106% to 77%). It can be concluded that HT29 cells were more sensitive than Bx-PC3 cells. Finally, both clonal and commercial wines serve as valuable sources of polyphenolic compounds, which could have a significant role in preventing cancer and diseases related to oxidative stress.

CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) was the first Italian protontherapy facility dedicated to the treatment of ocular neoplastic pathologies. It is in operation at the LNS Laboratories of the Italian Institute for Nuclear Physics (INFN-LNS) and to date, 500 patients have been successfully treated. Even though proton therapy has demonstrated success in clinical settings, there is still a need for more accurate models because they are crucial for the estimation of clinically relevant RBE values. Since RBE can vary depending on several physical and biological parameters, there is a clear need for more experimental data to generate predictions. Establishing a database of cell survival experiments is therefore useful to accurately predict the effects of irradiations on both cancerous and normal tissue. The main aim of this work was to compare RBE values obtained from in-vitro experimental data with predictions made by the LEM II (Local Effect Model), Monte Carlo approaches, and semi-empirical models based on LET experimental measurements. For this purpose, the 92.1 uveal melanoma and ARPE-19 cells derived from normal retinal pigmented epithelium were selected and irradiated in the middle of clinical SOBP of the CATANA proton therapy facility. The remarkable results show the potentiality of using microdosimetric spectrum, Monte Carlo simulations and LEM model to predict not only the RBE but also the survival curves.

Even though protons show several benefits for the therapy of ocular tumors, the application of a single RBE value in eye treatment planning is still questionable and requires further investigation. In this work, the effects of protons were experimentally measured on 92.1, a specific uveal melanoma cell line. The chosen irradiation position was the middle of the 62 MeV therapeutic Spread-Out Bragg Peak. The normal, ARPE-19 retinal cells were also used to assess the behaviour of healthy tissue exposed to irradiation. Cells were irradiated with single doses ranging from 1 to 6 Gy, and the clonogenic assay was adopted to evaluate cell survival after irradiation. The radiobiological results suggest that proton irradiation can cause significant lethal damages in both normal and cancerous cells. The obtained experimental data from biological models will serve for comparisons with Monte Carlo simulations and LEM II calculations.

Aim: Newly synthesized platinum(IV) complexes with ethylenediamine-N,N’-diacetate ligands (EDDA-type) (butyl-Pt and pentyl-Pt) were investigated against two cancer (A549 lung, and HTB 140 melanoma) and one non-cancerous (MRC-5 embryonic lung fibroblast) human cell lines. Materials and Methods: The effects of these agents were compared with those of cisplatin after 6-, 24- and 48-h treatment. Sulforhodamine-B (SRB) assay was performed to estimate the cytotoxic effect, while the inhibitory effect on cell proliferation was measured using 5-bromo-2,-deoxyuridine (BrdU) incorporation assay. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by these agents was determined by electrophoretic analysis of DNA, flow cytometry and by western blot analysis of proteins involved in induction of apoptosis. The effects of gamma irradiation, alone and in combination with platinum-based compounds, were examined by clonogenic and SRB assays. Results: All examined platinum-based compounds had inhibitory and antiproliferative effects on A549 cells, but not on HTB140 and MRC-5 cells. Butyl-Pt, pentyl-Pt and cisplatin arrested the cell cycle in the S-phase and induced apoptotic cell death via regulation of expression of B-cell lymphoma 2 (BCL2) and BCL2-associated X (BAX) proteins. Platinum-based compounds increased the sensitivity of A549 cells to gamma irradiation. Butyl-Pt and pentyl-Pt showed better antitumour effects against A549 cells than did cisplatin, by interfering in cell proliferation and the cell cycle, and by triggering apoptosis. Conclusion: The effects of gamma irradiation on tumour cells may be amplified by pre-treatment of cells with platinum-based compounds.

The aim of the present study was to investigate effects of pomegranate peel (PP) extract on different human cancer cell lines. MTT was performed to estimate cytotoxic effects of PP extract against HTB140, HTB177, MCF7, HCT116 human cancer cell lines and MRC-5 normal fibroblasts. Clonogenic assay was used to reveal cell survival after the treatment with PP extract. Cell cycle analysis was done using flow cytometry. Wound healing assay was applied to estimate inhibitory effects of PP extract on migration of cancer cells. The results showed that PP extract expressed selective cytotoxicity for cancer cells compared to normal cell line. Analyzed cancer cell lines displayed individual variations in sensitivity to PP extract reflected through changes in clonogenic survival, cell cycle distribution and migration, which may be due to the specific nature of each tested cell line. In conclusion, PP extract exhibits good inhibitory effects on tested cancer cell lines.

Purpose: Investigation of effects on DNA of γ-irradiated human cancer cells pretreated with free radical scavengers is aimed to create reference data which would enable assessment of the relative efficiency of high linear energy transfer (LET) radiations used in hadron therapy, i.e. protons and carbon ions. Materials and methods: MCF-7 breast and HTB177 lung cancer cells are irradiated with γ-rays. To minimize indirect effects of irradiation, dimethyl sulfoxide (DMSO) or glycerol are applied as free radical scavengers. Biological response to irradiation is evaluated through clonogenic cell survival, immunocytochemical and cell cycle analysis, as well as expression of proteins involved in DNA damage response. Results: Examined cell lines reveal similar level of radioresistance. Application of scavengers leads to the rise of cell survival and decreases the number of DNA double strand breaks in irradiated cells. Differences in cell cycle and protein expression between the two cell lines are probably caused by different DNA damage repair mechanisms that are activated. Conclusion: The obtained results show that DMSO and glycerol have good scavenging capacity, and may be used to minimize DNA damage induced by free radicals. Therefore, they will be used as the reference for comparison with high LET irradiations, as well as good experimental data suitable for validation of numerical simulations. © 2019, © 2019 Taylor & Francis Group, LLC.

The aim of this study was to investigate effects of irradiations with the therapeutic proton and carbon ion beams in two non-small cell lung cancers, CRL5876 adenocarcinoma and HTB177 large cell lung carcinoma. The DNA damage response dynamics, cell cycle regulation, and cell death pathway activation were followed. Viability of both cell lines was lower after carbon ions compared to the therapeutic proton irradiations. HTB177 cells showed higher recovery than CRL5876 cells seven days following the treatments, but the survival rates of both cell lines were lower after exposure to carbon ions with respect to therapeutic protons. When analyzing cell cycle distribution of both CRL5876 and HTB177 cells, it was noticed that therapeutic protons predominantly induced G1 arrest, while the cells after carbon ions were arrested in G2/M phase. The results illustrated that differences in the levels of phosphorylated H2AX, a double-strand break marker, exist after therapeutic proton and carbon ion irradiations. We also observed dose- and time-dependent increase in the p53 and p21 levels after applied irradiations. Carbon ions caused larger increase in the quantity of p53 and p21 compared to therapeutic protons. These results suggested that various repair mechanisms were induced in the treated cells. Considering the fact that we have not observed any distinct change in the Bax/Bcl-2 ratio following irradiations, it seemed that different types of cell death were involved in the response to the two types of irradiations that were applied.

Background and objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions (C-12) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon (C-12) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/mu m. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. Results: Cell viability experiments indicated strong anti-tumour effects of C-12 ions. The analysis of cell cycle showed that C-12 ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/mu m at the dose level of 16 Gy. Pro-apoptotic effects of C-12 ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NF kappa B). At the level of protein expression, the results indicated significant increases of p53, NF kappa B and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NF kappa B mRNA. Interpretation and conclusions: The present results indicated that anti-tumour effects of C-12 ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (gamma H2AX). Immunofluorescent staining visualizes formation of gamma H2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of gamma H2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to gamma-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of gamma H2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of gamma H2AX foci.

In most patients with lung cancer radiation treatment is used either as single agent or in combination with radiosensitizing drugs. However, the mechanisms underlying combined therapy and its impact on different modes of cell death have not yet been fully elucidated. We aimed to examine effects of single and combined treatments with gamma-rays and erlotinib on radioresistant CRL-5876 human lung adenocarcinoma cells with particular emphasis on cell death. CRL-5876 cells were treated with gamma-rays and/or erlotinib and changes in cell cycle, DNA repair dynamics, ultrastructure, nuclear morphology and protein expression were monitored at different time points. To reveal the relationship between types of cell death that arise after these treatments, autophagy was blocked with chloroquine. We found that higher dose of gamma-rays causes G2/M arrest while adding of erlotinib to this treatment decreases the number of cells in S phase. Impact of erlotinib on kinetics of disappearance of irradiation-induced DNA double strand breaks is reflected in the increase of residual gamma-H2AX foci after 24 h. gamma-rays provoke cytoprotective autophagy which precedes development of senescence. Erlotinib predominantly induces apoptosis and enlarges the number of apoptotic cells in the irradiated CRL-5876 cells. Chloroquine improved cytotoxicity induced by radiation and erlotinib, increased apoptosis and decreased senescence in the CRL-5876 cells. The results obtained on CRL-5876 cells indicate significant radiosensitizing effect of erlotinib and suggest that chloroquine in the combination with the above treatments may have an additional antitumor effect in lung adenocarcinoma.

Zračenje indukuje povišenu ekspresiju receptora za epidermalni faktor rasta (EGFR). Polazeći od značaja EGFR signalnog puta u procesu tumorogeneze, ovaj receptor je postao važan ciljani molekul u terapiji tumora. Utvrđeno je da je on pojačano eksprimiran i u nesitnoćelijskom karcinomu pluća i da ima važnu ulogu u patogenezi i prognozi terapijskog ishoda ove bolesti. Erlotinib, inhibitor tirozin kinazne funkcije EGFR je jedan od prvih agenasa koji su uvedeni u ciljanu terapiju nesitnoćelijskog karcinoma pluća. S obzirom na navedeno, istraživanja sinergističkog delovanja erlotiniba i zračenja u cilju poboljšanja antitumorskog efekta ovih agenasa su veoma aktuelna. Mehanizmi koji se nalaze u osnovi kombinovane terapije i njenog uticaja na nastanak različitih tipova ćelijske smrti su još uvek nedovoljno istraženi. U ovoj disertaciji je in vitro ispitivan antitumorski efekat pojedinačnih i kombinovanih tretmana γ-zračenjem i erlotinibom na CRL-5876 ćelije nesitnoćelijskog karcinoma pluća čoveka. Efikasnost pojedinačnih i kombinovanih tremana na ćelijsko klonogeno preživljavanje, vijabilnost, proliferaciju, kao i na kinetiku nastanka i nestanka dvolančanih prekida na DNK i distribuciju ćelija po fazama ćelijskog ciklusa, praćena je u funkciji vremena. U okviru ove studije analizirani su i efekti navedenih tretmana na promenu morfologije i ultrastrukture ćelija. U cilju utvrđivanja tipova ćelijske smrti do kojih ovi agensi dovode i njihovog međusobnog odnosa, uveden je inhibitor autofagije, hlorokin. Na osnovu radiobioloških parametara utvrđeno je da CRL-5876 ćelije adenokarcinoma pluća čoveka pripadaju grupi radiorezistentnih tumorskih ćelija. Promene radiobioloških parametara nakon kombinovanog tretmana ukazuju da erlotinib značajno povećava radioosetljivost CRL-5876 ćelija. Ove promene su praćene i na nivou ćelijskog ciklusa. Zračenje izaziva G2/M blok, dok erlotinib dovodi do smanjenja S faze..., It is known that radiation therapy increases EGFR expression in cancer cells, thus promoting radioresistence. Considering the importance of EGFR signaling pathway in tumorigenesis, this receptor has become an important target molecule for the treatment of tumors. It has been shown that EGFR is frequently overexpressed in non-small cell lung cancer (NSCLC) and plays an important role in the pathogenesis and the prediction of the therapeutic outcome of the disease. Erlotinib inhibits the tyrosine kinase activity of the EGFR and is used in molecular targeted therapy of NSCLC. Given the above findings, combining radiation with erlotinib in order to boost antitumor effects of these agents represents an actual field of research. However, the mechanisms underlying combined therapy and its impact on the induction of different modes of cell death have not been fully explained. This theasis examines the antitumor effect of single and combined treatments with γ-rays and erlotinib on CRL-5876 human lung adenocarcinoma cells in vitro. The efficiency of single and combined treatments on clonogenic survival, cell viability, proliferation, kinetics of formation and disappearance of DNA double strand breaks and cell cycle distribution is monitored as a function of time. In this study the effects of these treatments on cell morphology and ultrastructure are analyzed. In order to determine the modes of cell death caused by these agents and to reveal their mutual relations, the autophagy inhibitor chloroquine is introduced in the analysis. Based on the radiobiological parameters it is found that CRL-5876 human lung adenocarcinoma cells belong to the radiorezistant group of cells. Changes in radiobiological parameters after combined treatments show that erlotinib significantly increases radiosensitivity of CRL-5876 cells. Moreover, changes in cell cycle distribution after treatments are observed. It is revealed that radiation causes G2/M arrest while erlotinib induces a decrease in S ph

Introduction: Proton radiation offers physical advantages over conventional radiation. Radiosensitivity of human 59M ovarian cancer and HTB140 melanoma cells was investigated after exposure to gamma-rays and protons. Material and methods: Irradiations were performed in the middle of a 62 MeV therapeutic proton spread out Bragg peak with doses ranging from 2 to 16 Gy. The mean energy of protons was 34.88+/-2.15 MeV, corresponding to the linear energy transfer of 4.7+/-0.2 keV/mu m. Irradiations with gamma-rays were performed using the same doses. Viability, proliferation and survival were assessed 7 days after both types of irradiation while analyses of cell cycle and apoptosis were performed 48 h after irradiation. Results: Results showed that gamma-rays and protons reduced the number of viable cells for both cell lines, with stronger inactivation achieved after irradiation with protons. Surviving fractions for 59M were 0.91+/-0.01 for gamma-rays and 0.81+/-0.01 for protons, while those for HTB140 cells were 0.93+/-0.01 for gamma-rays and 0.86+/-0.01 for protons. Relative biological effectiveness of protons, being 2.47+/-0.22 for 59M and 2.08+/-0.36 for HTB140, indicated that protons provoked better cell elimination than gamma-rays. After proton irradiation proliferation capacity of the two cell lines was slightly higher as compared to gamma-rays. Proliferation was higher for 59M than for HTB140 cells after both types of irradiation. Induction of apoptosis and G2 arrest detected after proton irradiation were more prominent in 59M cells. Conclusions: The obtained results suggest that protons exert better antitumour effects on ovarian carcinoma and melanoma cells than gamma-rays. The dissimilar response of these cells to radiation is related to their different features.

Molecular targeted cancer therapy is a promising treatment strategy. Considering the central role of the epidermal growth factor receptor in cell proliferation and survival, there are indications that targeted agents like tyrosine kinase inhibitors, i. e., erlotinib, may enhance the antitumor treatment by radiation. The aim of this study is to analyze the inactivation effects of gamma-rays and to test the radiosensitizing potential of erlotinib on human lung adenocarcinoma cells in vitro. Irradiations were performed with doses ranging from 1 Gy to 8 Gy. In order to increase the radiosensitivity of CRL-5876 lung adenocarcinoma cells, the cells were treated with a clinically relevant concentration of 2 mu M erlotinib. The effects of single and combined treatments were monitored using clonogenic survival, cell viability and proliferation assays at different time points. For the detection and visualization of the phosphorylated histone H2AX (gamma-H2AX), an important biological marker of DNA double-strand break formation, fluorescence inununocytochemistry, was performed. The response to the treatment was monitored at four time points: 30 min, 2, 6, and 24 h. Irradiations with gamma-rays resulted in significant cell inactivation regarding all analyzed biological endpoints. Combined treatments revealed consistent cell inactivation. Moreover, compared to gamma-rays alone, elevated levels of gamma-H2AX foci were observed after pretreatment with erlotinib, indicating radiosensitization through impaired DNA repair.

The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumour cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (gamma-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (gamma-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of gamma-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between gamma-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the database that might promote pulsed sources for medical treatments of malignant growths.

This study was conducted in order to evaluate the ability of carbon ions to induce DNA double-strand breaks and apoptosis in the radio-resistant human HTB140 melanoma cells. The cells were irradiated with C-12 ions having the linear energy transfer of 258 keV/mu m. Irradiations were performed in the dose range from 2 to 16 Gy. Induction of DNA double-strand breaks was evaluated 2 hour after irradiation through expression of gamma H2AX protein. Increased level of gamma H2AX detected in irradiated samples was especially high after irradiation with 12 and 16 Gy. Dose dependent increase of apoptosis was detected 48 hour after irradiation by flow-cytometry, with the maximum value of 20.4% after irradiation with 16 Gy, and the apoptotic index of 9.3. Pro-apoptotic effects of carbon ion beams were confirmed by changes of key molecules of the mitochondrial apoptotic pathway, p53 protein expression, Bax/Bcl-2 ratio and caspase-3 activation.

The effects of γ-rays on the DNA level, i.e. formation of double-strand breaks and expression of p21 were studied in vitro on the human HTB 140 melanoma cells. Cells were exposed to the dose range from 2 to 16 Gy. Effects were analyzed 30 min, 2, 6 and 24 h after irradiation. It has been shown that the level of phosphorylated histone H2AX (γH2AX) is time- and dose-dependent, as well as the expression of p21.

In order to increase radio-sensitivity of human lung adenocarcinoma NCI-H1568 cells, targeted therapy drug, erlotinib was used. The impact of radiation and erlotinib on cell behaviour was analyzed using three biological endpoints. Irradiations with γ-rays resulted in reduction of cell survival, viability and proliferation. Erlotinib significantly inhibited cell growth and proliferation capacity. Combined treatments with radiation and erlotinib showed high level of reduction of cell viability and proliferation. Preliminary data encourage further investigations of mechanisms underlying the radiation responses enhanced by erlotinib.

Response of human HTB140 melanoma cells to proton irradiation in combination with fotemustine (FM) was investigated. Effects of these agents were analysed on cell proliferation and induction of apoptosis. Cells pretreated with 100- or 250-mu M of FM were irradiated in the middle of the therapeutic 62-MeV proton spread-out Bragg peak, with a dose of 16 Gy. All treatments reduced proliferation and survival of melanoma cells. The most pronounced effects of the combined treatment were obtained for cell survivals. The level of apoptosis increased after all applied treatments. Particularly good pro-apoptotic effect was achieved when proton irradiation was combined with 250 mu M of FM. This was followed by the increased expression of p53 gene. The obtained results have shown that combined application of FM and protons significantly reduced growth of this resistant melanoma cell line.

Conventional radiotherapy with X-and gamma-rays is one of the common and effective treatments of cancer. High energy hadrons, i.e., charged particles like protons and (12)C ions, due to their specific physics and radiobiological advantages are increasingly used. In this study, effectiveness of different radiation types is evaluated on the radio-resistant human HTB140 melanoma cells. The cells were irradiated with gamma-rays, the 62 MeV protons at the Bragg peak and in the middle of the spread-out Bragg peak (SOBP), as well as with the 62 MeV/u (12)C ions. The doses ranged from 2 to 24 Gy. Cell survival and proliferation were assessed 7 days after irradiation, whereas apoptosis was evaluated after 48 h. The acquired results confirmed the high radio-resistance of cells, showing better effectiveness of protons than gamma-rays. The best efficiency was obtained with (12)C ions due to higher linear energy transfer. All analyzed radiation qualities reduced cell proliferation. The highest proliferation was detected for (12)C ions because of their large killing capacity followed by small induction of reparable lesions. This enabled unharmed cells to preserve proliferative activity. Irradiations with protons and (12)C ions revealed similar moderate pro-apoptotic ability that is in agreement with the level of cellular radio-resistance.

Response of human HTB140 melanoma cells to proton irradiation in combination with fotemustine (FM) was investigated. Effects of these agents were analysed on cell proliferation and induction of apoptosis. Cells pretreated with 100- or 250-mu M of FM were irradiated in the middle of the therapeutic 62-MeV proton spread-out Bragg peak, with a dose of 16 Gy. All treatments reduced proliferation and survival of melanoma cells. The most pronounced effects of the combined treatment were obtained for cell survivals. The level of apoptosis increased after all applied treatments. Particularly good pro-apoptotic effect was achieved when proton irradiation was combined with 250 mu M of FM. This was followed by the increased expression of p53 gene. The obtained results have shown that combined application of FM and protons significantly reduced growth of this resistant melanoma cell line.